Use of inhibitors for the treatment of RTK-hyperfunction-induced disorders, particularly cancer

ABSTRACT

The present invention concerns the use of inhibitors for the treatment and/or prophylaxis of diseases which are the consequence of increased receptor tyrosine kinase activity, particularly cancer. The use is particularly directed towards inhibition or lowering of the overexpression and/or altered activity of receptor tyrosine kinases (RTKs). In particular, this altered activity of receptor tyrosine kinase can be triggered by a mutation of FGFR-4, wherein this mutation is in particular a point mutation in the transmembrane domain of FGFR-4 and leads to an exchange of a hydrophobic amino acid for a hydrophilic amino acid. The invention further concerns the use of an inhibitor directed against FGFR-4, for the treatment and/or prophylaxis of cancer. Furthermore, the invention concerns a mutated FGFR-4, which leads to overexpression and/or altered activity in cells. Finally, the invention concerns a DNA and RNA sequence of a mutated FGFR-4 molecule. Finally, in addition the invention concerns a pharmaceutical composition, containing the inhibitor as described above and further a diagnostic and screening procedure.

[0001] The present invention concerns the use of inhibitors for thetreatment and/or prophylaxis of diseases which are the consequence ofincreased receptor tyrosine kinase activity, particularly cancer. Theuse is particularly directed towards inhibition or lowering of theoverexpression and/or altered activity of receptor tyrosine kinases(RTKs). In particular, this altered activity of receptor tyrosine kinasecan be triggered by a mutation of FGFR-4, wherein this mutation is inparticular a point mutation in the transmembrane domain of FGFR-4 andleads to the exchange of a hydrophobic amino acid for a hydrophilicamino acid. The invention further concerns the use of the inhibitors ofFGFR kinases, particularly for the treatment and/or prophylaxis ofcancer. Furthermore, the invention concerns a mutated FGFR-4, whichleads to overexpression and/or altered activity in cells. Finally, theinvention concerns a DNA and RNA sequence of a mutated FGFR-4 molecule.Finally, in addition the invention concerns a pharmaceuticalcomposition, containing the inhibitor as described above and, further, adiagnostic and screening procedure.

[0002] Cell growth is a carefully regulated process dependent on thespecific needs of an organism. In a young organism, the cell divisionrate exceeds the cell death rate, which leads to an increase in the sizeof the organism. In an adult organism, the new formation of cells andcell death are balanced so that a “steady state” arises. In rare cases,however, the control of cell multiplication breaks down and the cellsbegin to grow and to divide, although no specific need for a highernumber of cells of this type exists in the organism. This uncontrolledcell growth is the cause of cancer. Factors that can provoke theuncontrolled cell growth, sometimes associated with metastasisformation, are often of a chemical nature, but can also be of a physicalnature, such as for example radioactive radiation. Another cause of thetriggering of cancer are genetic peculiarities or mutations in a certainorganism, which sooner or later lead to the cells degenerating.

[0003] Up to now, it has still not been possible satisfactorily toelucidate the processes which control normal growth and differentiation,for example in the breast. In addition to hormonal control, there isalso a complex network of different, locally generated growth factorswhich intervene in the development of the mammary cells. The precisecauses of the occurrence of cancer in mammary cells are as unclear andunknown as they are diverse, as is also the case with other cells.Alterations in oncogenes and tumour suppressor genes appear to play animportant part in breast cancer carcinogenesis. In addition, reinforcedstimulation by regulatory factors which arise in genetically alteredcells can lead to increased progression of cell growth.

[0004] At present, essentially two alternatives are available for thetreatment of cancer. Either the cancer cells are successfiully removedfrom the diseased organism completely by a surgical intervention, orattempts are made to render the degenerated cells in the organismharmless, for example by administration of medicaments (chemotherapy) orby physical therapeutic procedures, such as irradiation.

[0005] In chemotherapy, medicaments are often used which in some formintervene in the DNA metabolism and damage rapidly growing cells, whichhave to produce higher DNA metabolic capacity, more strongly than cellswhich are dividing slowly or not at all. However, a severe disadvantageof many chemotherapeutic drugs is the low specificity of the activesubstance used, as a result of which healthy cells are also damagedduring the chemotherapy. This low specificity of the active substancesfurther requires that their dosage must in each case be such that as fewas possible healthy cells are damaged, with simultaneous killing of thecancer cells. This is often not possible, and the cancer patient diesbecause of the ever further spreading cancer cells, which in the finalstages cause the failure of vital functions.

[0006] It is assumed that the overexpression and/or altered activity ofcertain growth factor receptors contribute to the intensified growth ofmany neoplasms, including breast cancer. For example, the overexpressionof EGFR, i.e. epidermal factor receptor, or ERB B-2 receptor in breasttumours has been linked with a poor prognosis. FGF (historically:fibroblast growth factor) proteins could also be involved in thedevelopment of cancer in breast glands or of other cancer; however theresults in this regard are contradictory or are inconclusive.

[0007] The FGFs constitute a large family of peptide regulatory factors,of which 9 members are so far known. Eight of these have been wellcharacterised in man (Basilico and Moscatelli, 1992; Coulier et al.,1993). The FGFs operate via high-affinity tyrosine kinase receptors,which are coded for by at least four different genes. Further, the FGFsare multifunctional, regulatory peptides wnich could have an effect notonly on tumorigenesis but could also play a major part in cardiovasculardiseases, reconstruction after tissue injury, neurobiology and embryonicdevelopment. The acidic and basic FGFs (AFGF and bFGF) were the firstand are the best characterised members of the family. In vivo it couldfor example be shown that FGFs are involved in mesodermal induction inembryogenesis (Slack et al., 1987; Kimelman et al., 1988), and alsoinvolvement in angiogenesis (Thomas et al., 1985; Thompson et al., 1989;Folkmann and Klagsbrun, 1987).

[0008] For the corresponding receptors (FGFRs), four similar genescoding for them have been identified. These genes code for structurallyrelated proteins with an extracellular domain which consists of threeimmunoglobulin loops and an acid portion, a hydrophobic trans membranedomain and an intracellular domain, which incorporates a tyrosine kinaseactivity. For two of these genes, FGFR-1 and FGFR-2, it could be shownthat they have multiple transcripts, which arise by alternative splicing(Givol and Yayon, 1992 and Johnson and Wllliams, 1993). Splice variantswhich arise from these genes differ with respect to the number ofimmunoglobulin-like domains in the extracellular region of the receptorand in the sequence for the second half of the third immunoglobulindomain, which can arise from alternative exons. In addition,transmembrane and juxtamembrane shortenings or deletions can arise,which can generate secreted or kinase-inactive protein products.

[0009] For FGFR-3, it was possible to find alternative transcripts andcorresponding isoforms, but for FGFR-4 there is only a single knownprotein product. Because of the large number of FGFR genes andtranscripts and the lack in many protein products of a specificity fordefined FGFs, it is difficult to determine the action of a specificligand on a specific receptor. Hence, correlations between specific FGFreceptors and defined diseases can only be established with greatdifficulty, let alone a correlation of a particular mechanism of actionof a defined receptor with a disease. Accordingly, it is difficulteffectively to treat diseases, especially the complex disease picturecancer, utilising the FGFRs.

[0010] Hence it is an objection of the present invention to specify apossible treatment and/or prophyl-axis of somatic disorders, in thedevelopment of which receptor tyrosine kinases (RTKs) are involved,particularly cancer. In particular, it is an objection of the presentinvention to inhibit and/or to lower overexpression and/or altered, forexample constitutive activity of receptor tyrosine kiases.

[0011] It is further an objection of the present invention to inhibitand/or to lower the altered activity of the receptor tyrosine kinase ofa mutated FGFR-4.

[0012] It is a further an objection of the present invention to specifya further RTK which is involved in carcinogenesis and/or metastasisformation. Further, it is an objection of the present invention tospecify a DNA sequence or corresponding RNA sequence of the RTK.

[0013] It is a further an objection of the present invention to specifyimproved diagnostic or differential diagnostic and screening procedures.

[0014] Finally it is an objection of the present invention to specify apharmaceutical composition, with which in particular cancer can betreated.

[0015] These objections are achieved by the objects of the independentclaims. The dependent claims specify preferred developments of theinvention.

[0016] For the better understanding of the present invention, the termsused herein are explained in more detail.

[0017] By “inhibitor” is understood any substance which inhibits the RTKor lowers their activity. This can be a low-molecular weight substancedirected against the RTK, a kinase-inactive receptor or an anti-receptorantibody.

[0018] By “kinase-inactive receptors is understood any receptor which nolonger has any tyrosine kinase activity.

[0019] By “receptor tyrosine kinase” [sic] is understood any receptorwhich has tyrosine kinase activity. The expression includes growthfactor receptors which have tyrosine kinase activity, and also HER2 orthe met-receptors.

[0020] By “RTK-Hyperfunction” is understood overexpression (see below)and/or altered activity (see below).

[0021] “Defective signal transfer activities” means that a mutatedreceptor is no longer capable of converting an extracellular growthsignal or another signal into an intracellular signal, in the sense thatthis defective signal generation no longer depends on the presence of aligand, for example the growth factor.

[0022] “Growth factor” means any mitogenic chemical, usually apolypeptide, which inter alia is secreted by normal and/or transformedmammalian cells, and which plays a significant part in the regulation ofcell growth, in particular in the stimulation of the proliferation ofthe cells and the maintenance of their viability. The term “growthfactor” for example includes epidermal growth factor (EGF),platelet-derived growth factor (PDGF) and nerve growth factor (NGF), andalso FGF, namely fibroblast growth factor.

[0023] By “mutated receptor tyrosine kinase” is understood a receptortyrosine kinase which by comparison with the wild type receptor containsa structural alteration, so that the receptor has a different, e.g. nolonger regulable, tyrosine kinase activity from the wild type receptor.One class of mutations leads to altered activity of the RTK.

[0024] By wild type growth factor receptor” or “wild type” receptor isunderstood a naturally occurring growth factor receptor or receptor thatbears the non-mutated amino acid sequence. The “wild type” correspondsto the receptor variant most commonly occurring in the population.

[0025] By “extracellular domain” of the growth factor receptor orreceptor is understood the part of the receptor which normally projectsout of the cell into the extracellular surroundings. The extracellulardomain for example includes the part of the receptor to which a growthfactor or another molecule (ligand) binds.

[0026] By “transmembrane region” of the growth factor receptor orreceptor is understood the hydrophobic portion of the receptor, which isnormally located in the cell membrane of the cell which expresses thereceptor.

[0027] By “tyrosine kinase domain” or “cytoplasmic domain” of the growthfactor receptor or receptor is understood the portion of the receptorwhich is normally situated inside the cell, and brings about thetransphosphorylation of tyrosine residues.

[0028] By “an effective quantity” is understood a quantity of thecomposition according to the invention which can achieve the desiredtherapeutic effect.

[0029] By “fibroblast growth factor (FGF)” is understood a mitogenicpolypeptide which influences the growth and other properties of cells,inter alia of fibroblasts.

[0030] By “overexpression” is understood increased production of RTKprotein by a cell as compared to the wild type. This can for example betriggered by gene amplification of the RTK gene and lead to excessive,uncontrolled cell division activity.

[0031] By “altered activity” is understood permanent activity of asignal transfer route mediated by growth factor receptors. Thus with analtered RTK the knase activity is also present when no ligand ispresent.

[0032] According to the present invention, it could be shown that amutated FGFR-4 can lead to overexpression and/or altered activity of thecorresponding receptor tyrosine kinase in cells and hence lead tocancer.

[0033] Growth factor receptors play a decisive part in the developmentand multiplication of human cancer cells. In healthy cells, the growthfactor receptors are inter alia involved in the control of cell growth,but also in differentiation, cell migration, etc. The actual signal forthe cell division is the growth factor, which is formed depending on theneeds of the organism. The receptor undertakes the function of signaltransfer, i.e. it is involved in the conversion of the extracellulargrowth signal into cell division activity in the inside of the cell.With many growth factor receptors, their ability, after binding of thegrowth factor to the extracellular domain, to transfer phosphateresidues onto tyrosine residues in proteins plays a decisive part. Thesereceptors are also described as receptor tyrosine kinases. A review ofreceptor tyrosine kinases is to be found in Yarden Y and Ullrich A, Rev.Biochem. 1988, 57, 443-78. The dimerisation of these growth factorreceptors after binding of the growth factor is a further importantevent in the process of signal transfer. The conversion of anextracellular signal into an intracellular signal mediated by growthfactor receptors with tyrosine kinase activity can be broken down intothe following five steps:

[0034] 1. The binding of the growth factor (also described as ligand) tothe extracellular domain of the receptor induces a conformationalchange; this causes

[0035] 2. dimerisation of receptors with altered conformation; with

[0036] 3. simultaneous induction of kinase activity;

[0037] 4. transphosphorylation of tyrosine residues in the receptordimer, which once again creates and stabilises an activated receptorconformation; and

[0038] 5. phosphorylation of polypeptide substrates and interaction withcellular factors.

[0039] Uncontrolled hyperfunction of this signal transfer chain forexample because of the overexpression or altered activity of thereceptor can inter alia lead to increased division activity of therelevant cells and in the extreme case to a degenerated cancer cell. Areview concern-ing growth factor receptors and their function in signaltransfer from the extracellular to the intracellular milieu, and thepossible influence of abnormally expressed receptors on carcino-genesis,is given in Ullrich A and Schiessinger J (1990) Cell 61, 203-212.

[0040] It has now surprisingly been found that in the five-stage signaltransfer chain explained above, a mutated FGFR-4 results in increasedsignal transfer activity, in the development of which the alteredactivity of mutated RTK is decisively involved.

[0041] Hence according to claim 1 of the present invention at least oneinhibitor of a receptor tyrosine kinase is used for the treatment and/orprophylaxis of RTK-hyperfunction-induced disorders, particularly cancer.Furthermore, according to the invention diseases or somatic disorderswhich are the consequence of a hyperproliferation of tissues and/orincreased invasivity of tissues attributable to increased signaltransfer can also be eliminated or alleviated.

[0042] As inhibitor, as well as low-molecular weight substances, forexample at least one kinase-inactive receptor can be used. Through theuse of the inhibitor, e.g. of the kinase-inactive receptor, the-alteredactivity of the receptor tyrosine kinase can be inhibited and/orlowered. As has already previously been stated, the overexpressionand/or altered activity of growth factor receptors is an importantfactor in the triggering or the progression of cancer. Theoverexpression of EGFR or the Erb B-2 receptor in breast tumours has forexample been associated with a poor prognosis (see above). Henceinhibition of this overexpression and/or altered activity is animportant component in the treatment and/or prophylaxis of cancer.FGFR-4 is tissue-specifically switched off during embryogenesis.However, it is present in 30% of breast cancer patients; it is notdetectable in the tissue of healthy subjects. The use of inhibitors forreceptor tyrosine kinase leads to a lowering or complete inhibition ofthe overexpression and/or altered activity. Likewise, the use ofkinase-inactive receptors leads to a lowering and/or complete inhibitionof the activity of the receptor tyrosine kinases, since the kinasefunction of the heterodimer is no longer capable of signal transfer. Theaction of kinase-inactive receptors is based on the fact thatnon-functional heterodimers are formed (dilution effect). A lack ofsignal transfer leads to prevention of the transmission of theoverexpressed and/or altered active signal, as a result of which thesignal is prevented from conversion into a biological response of thecell. As a result, through this inhibition of the receptor tyrosinekinase or through these kinase-inactive receptors, it is possibleeffectively and positively to intervene in the treatment and/orprophylaxis of cancer.

[0043] It has surprisingly been found that the FGFR-4 mutation alsooccurs in the germ line of healthy persons. It is assumed that the germline mutation leads to a genetic predisposition, which renders thepersons concerned susceptible to the outbreak of various diseases. Inconnection with carcinogenesis, it is assumed that the increaseexpression of the mutated receptor in the tumour tissue is involved inthe carcinogenesis. The germ line mutation is further regarded as apredisposition inter alia for the following diseases: arteriosclerosis,leukaemia, lymphoma, hepatic cell carcinoma and cholangiocarcinoma.

[0044] Consequently, the present invention makes a further geneticmarker available, which is found to be extremely helpful in thediagnosis and early recognition of various diseases and susceptibilityto these.

[0045] The present invention therefore also concerns a procedure for thedetection of a nucleic acid which codes for FGFR-4 in case material,whereby in particular mutations of the receptor-coding nucleic acid aredetected. This can for example be effected by hybridisation witholigonucleotide probes, which can specifically indicate the presence orabsence of a mutation, in particular a point mutation. In this, forexample a “mismatch” between mutated nucleic acid and oligonucleotide isutilised such that if a “mismatch” is present a hybridisation does nottake place and hence there is no signal. Alternatively, mutations canalso be detected by amplification of the nucleic acid with specificFGFR-4 PCR primers and subsequent cleavage with suitable restrictionendonucleases. If for example a mutation affects the recognitionsequence of a restriction endonuclease, such that for example themutated recognition sequence is no longer recognised as a cleavage siteby the restriction endonuclease, this leads to a different restrictionfragment than in the non-mutated wild type. By means of the PCR,restriction fragments can be specifically detected, so that in thestated case for example a larger restriction fragment is present in themutant compared to the wild type. Alternatively, however, a mutation canalso lead to the creation of a new restriction cleavage site, as aresult of which a “wild-type fragment” after cleavage with theappropriate enzyme becomes smaller in the mutant. The mutation in thetransmembrane domain of FGFR-4, at position 388 of the sequence, asdeposited in the EMBL Gene Bank/DDBJ under X57205, which leads to anexchange of Gly in the wild type for Arg in the mutant, concerns therecognition sequence GGWCC of the restriction endonuclease BstNl. As aresult, two new restriction fragments of 80 and 29 b.p. are formed,which can inter alia be detected by restriction analysis.

[0046] According to the present invention, it could further be shownthat overexpression and in particular altered activity of the RTK leadsto increased invasivity, i.e. to intensified metastasis formation. Sincemetastasis formation is one of the main problems of cancer, this meansthat the inhibition or lowering of the overexpression and/or alteredactivity will lead to an effective agent in the combating of cancer, bywhich in particular metastasis formation is inhibited.

[0047] Possible inhibitors are for example described in Mohammadi et al.(1997).

[0048] Preferably according to the invention an intervention is madeinto an overexpression and/or altered activity of the receptor tyrosinekinase, which is triggered by a mutation of FGFR-4. This mutation can beone or several point mutations. In particular, the mutation/mutationsoccur in the transmembrane domain of FGFRA, as a result of which inparticular a hydrophobic amino acid is exchanged for a hydrophilic aminoacid.

[0049] It is already known that point mutations which have led to anexchange of hydrophobic for hydrophilic amino acids in FGFR-3 areassociated with certain diseases. Thus for example, an altered activityof fibroblast growth factor receptor 3 due to a point mutation in thetransmembrane domain has been found in achondroplasia. Achondroplasia,which is the most commonly occurring genetic form of dwarfism, is anautosomal dominant disorder, which is essentially based on a defect inthe maturation process of certain bones. It could be shown thatachondroplasia is triggered by a Gly to Arg substitution in thetransmembrane domain of FGFR-3. It could further be shown that that theArg mutation in FGFR-3 activates the kinase function of the dimericreceptor. The Arg point mutation also leads to a ligand-independentstimulation of the tyrosine kinase activity of FGFR-3 itself and tostrongly increased altered levels of phosphotyrosine on the receptor.These results suggest that the molecular basis of achondroplasia isunregulated signal transfer by FGFR-3.

[0050] A further mutation in the transmembrane domain of FGFR-3 is anexchange alanine for glutamine. This amino acid exchange leads toanother disease, namely to Crouzon's disease with acanthosis nigricans.

[0051] According to the present invention, it was established thatmutations in FGFR, especially point mutations in the taansmembranedomain, which lead to an exchange of a hydrophobic for a hydrophilicamino acid, are involved in the triggering and poor prognosis forcancer, on account of which inhibition of receptor tyrosine kinases orthe use of kinase-inactive receptors are suitable for the treatmentand/or prophylaxis of cancer, wherein the receptor tyrosine kinases areoverexpressed or active in an altered way owing to a mutation.

[0052] In particular, for the point mutation at position 388, whichleads to an exchange of glycine for arginine, it could be shown that asa result of this the receptor tyrosine kinases become active in analtered way, and this homo- and heterozygotically results in signaltransfer without ligand stimulation, as a result of which in turn anuncontrollable growth of cells can be triggered. In the worst case, thisuncontrolled growth leads to cancer. The transmembrane domain then hasthe sequence (ID No. 1):

[0053] RYTDIILYASGSLALAVLLLLARLY,

[0054] while the non-mutated domain has the following sequence (IDNo.2):

[0055] RYTDIILYASGSLALAVLLLLAGLY.

[0056] Without being bound to one theory, it is assumed that theactivation of the receptor tyrosine kinase which bears one of theaforesaid point mutations and in particular the point mutation atposition 388, which leads to an exchange of glycine for arginine, isbased on a stabilisation of the receptor in a dimeric conformation,which occurs because of interactions through which changes in thetransmembrane domain were made possible. The intensified formation of aligand-independent dimer leads to increased receptor tyrosine kinaseactivity and cellular transformation. Other possibilities for the effectof the point mutations on the triggering of cancer may for example havea basis in that the mutation acts on the signal transfer by FGFR-4, inthat the receptor migration through the membrane is prevented, thereceptor dimerisation with itself or with other FGFRs is disturbed, orin that the tyrosine kinase activity of the receptor is affected.

[0057] According to the present invention, it could be shown that 56% ofpatients from St Petersburg with breast tumours (study of biopsies)carried the mutation at position 388, which is linked with an exchangeof glycine for arginine. Of these, 45% were heterozygotic and 11%homozygotic. This significantly high proportion suggests a link betweenthe point mutation at position 388 and the occurrence of breast cancer.

[0058] In a further study, in which German patients with breast tumourswere studied, only 43% of the patient showed the point mutations atposition 388. From the study with normal tissues of cancer patients andDNA from the tissue of normal individuals, it can be inferred that themutation is a germ line mutation.

[0059] Furthermore, genomic DNA and cDNA from cell lines was alsostudied, in order to determine the proportion of point mutations atposition 388. The cell lines studied derived from breast tumours, normalbreast epithelial cell lines as a comparison, squamous cell carcinoma,glioblastomas, neuroblastomas and uterine cancer. With all cell lines,except for the normal breast epithelia cell lines, a significantpercentage of the point mutation at position 388 in the FGFR-4 moleculecould be found. Hence the above-mentioned use of inhibitors orkinase-inactive receptors is especially suitable for the treatment ofcarcinomas. Here the treatment of neuroblastomas, uterine cancer andpancreatic cancer, but also other types of cancer, seems especiallypromising.

[0060] Particularly preferred is the use of inhibitors which inhibit amutated FGFR-4, especially with the mutation Gly→Arg at position 388 inthe transmembrane domain.

[0061] Further, the present invention concerns a mutated FGFR-4, whichleads to overexpression and/or altered activity of the receptor incells. Preferably, this mutated FGFR-4 is characterized in that ahydrophobic amino acid in the wild type receptor has been exchanged fora hydrophilic amino acid in the mutated receptor. Especially preferredis a mutation which is a point mutation and occurs in the transmembranedomain. Still more preferably, the point mutation occurs at position388, as a result of which preferably a glycine is replaced by arginine.

[0062] Hitherto, it was assumed among experts that only one FGFR-4occurs, which is not mutated. Mutated FGFR-4 was unknown. It wastherefore surprising that it could be shown according to the presentinvention that a mutated FGFR-4 exists. In particular, according to thepresent invention aconnection between the mutations, in particular thepoint mutation at position 388, and the occurrence of cancer could bedemonstrated. Furthermore, the germ line mutation in healthy persons hasbeen connected with the genetic predisposition for the occurrence interalia of arteriosclerosis.

[0063] The invention further concerns a DNA molecule containing asequence which codes for a mutated FGFR-4. The invention also includesan RNA molecule, containing an RNA sequence which codes for a mutatedFGFR-4. The above sequences can be used for diagnosis of cancer. Inthis, the sequences can specifically recognise the mutations in theFGFR-4. The presence of the mutation in the FGFR-4 is linked with a poorprognosis for the treatment of the cancer. The reason for this could beaggressive growth behaviour of the corresponding tumour.

[0064] Apart from this, the invention concerns a procedure for thedifferential diagnosis of breast cancer, wherein the patient's nucleicacid is brought into contact with one of the DNAs and/or RNAs describedabove, so that a signal is obtained, which indicates the presence and/orabsence of mutated FGFR-4. Finally, the present invention concerns apharmaceutical composition, containing the inhibitor or thekinase-inactive receptor, as described above. Apart from this, theinvention concerns a screening procedure for the identification ofinhibitors of tyrosine kinase activity, wherein the receptor accordingto the invention is brought into contact with potential inhibitors andthe tyrosine kinase activity in the presence and/or absence of theinhibitor is determined.

[0065] Further, the detection of the presence of a mutation can also beperformed by PCR and subsequent restriction enzyme cleavage, as alreadydescribed in more detail above.

[0066] Finally, other molecular biological diagnostic procedures arealso a possibility. Further, the object of the invention is an antibodywhich specifically reacts with a mutated FGFR-4 according to theinvention. “Specific” in the sense of the invention means that theantibody according to the invention binds to the mutated, but not to thenon-mutated, receptor.

[0067] Below, the invention is described in detail by the figures andexamples.

[0068] Here:

[0069]FIG. 1 shows SDS PAGE of an immunoprecipitation of FGFR-4 (thephosphorylated FGF receptor4 is marked by an arrow),

[0070]FIG. 2 a polyacrylamide gel of the mutated FGFR-4,

[0071]FIG. 3 a sequence analysis of the transmembrane domain of FGFR-4,

[0072]FIG. 4 the correlation between the FGFR-4 mutation G388R and thelymph node metastasis formation status (n=number of patients, p=P value)and

[0073]FIG. 5 the correlation between the FGFR-4 mutation G388R and therelapse-free survival time (n=number of patients, p=P value).

EXAMPLES

[0074] Cell Culture. The human cell lines MDA-MB453, ZR 75-1, K562 andSKBr3 were obtained from the ATCC. The individual supply sources can befound in the table at the end. MDA-MB453, K562 and ZR 75-1 werecultivated in RPMI (Gibco, Eggenstein) containing 10% foetal calf serum(Sigma, Taufkirchen). SKBR3 was cultivated in McCoy's 5a (Gibco,Egenstein) containing 15% foetal calf serum. All cell culture mediacontained penicillin/streptomycin (Sigma, Taufkirchen). The cells wereincubated at 37° C. in a water-vapour saturated atmosphere and 8% C02.

[0075] Cloning of FGFR-4^(388Arg)/wt. For preparation of RNA from K562and MDA-MB453 cells, 3×10⁷ cells were lysed with guanidiniumisothiocyanate and purified by ultracentrifugation in a CsCI gradient.The cDNA synthesis was effected with reverse transcriptase (Boehringer,Mannheim) and 10 pmol of “random oligonucleotides” in each case,according to the manufacturer's instructions. 0.5 pil were used in asubsequent PCR reaction.

[0076] FGFR-4^(Arg) and FGFR-4 wt were amplified by the PCR reaction.For this, the following primers were used:sense-GCTCAGAGGGCGGGCGGGGGTGCCGGCCG; anti-senseCCGCTCGAGTGCCTGCACAGCCTTGAGCCTTGC. For the PCR reaction, the followingwere used: 1.5 U/25 μl Expand-Polymerase (Boehringer, Mamnheim) andreaction buffer according to the manufacturer's instructions: 200 μMdNTP's; 0.01% v/v Triton X100; 10% v/v DMSO, and 0.2 pmol each of senseand a-sense primer. The following reaction steps were performed: 35cycles, 94° C. 1 min, 64° C. 1 min, 72° C. 2.5 min. MDA-MB453 cDNA wasused for the cloning of FGFR-4^(388Arg), and K562 cDNA for the cloningof FGFR-4 wt. The PCR products were cloned in the pcDNA3 vector(Invitrogen). In this way, both a FGFR-4 with the G388R and also a wildtype FGFR-4 could be obtained for further tests.

[0077] Amplification of the transmembrane domain of FGFR-4. Thefollowing primers were used: sense-GACCGCAGCAGCGCCCGAGGCCAG; anti-senseAGAGGGAAGAGG GAGAGCTTCTG. For the PCR reaction, the following were used:1.5 U/25 μl Taq-Polymerase (Boehringer, Mannheim) and reaction bufferaccording to the manufacturer's instructions: 200 μM dNTP's; 0.2 pmoleach of sense and a-sense primer, 0.5 μl cDNA or genomic DNA from tumourbiopsies and cell lines; the following reaction steps were performed: 35cycles, 95° C. 45 secs, 72° C. 45 secs.

[0078] Analysis by restriction digestion. The transmembrane domain ofFGFR-4 from genomic or cDNA was amplified as described above. To testbiopsies and cell lines for the G1217A mutation by restrictiondigestion, the PCR products were incubated for 1 hr at 60° C. with 5U/25 μl of BstNi (NEB, Schwalbach/Taunus). The DNA fragments from therestriction digestion were separated with a 20% polyacrylamide gel andstained with ethidium bromide. The analysis of the wild type receptoryields a 109, a 37 and a 22 base-pair sized fragment (track 4). On theother hand, as a result of the mutation G1217A a further restrictioncleavage site for BstN1 is formed. The mutated receptor shows further 80and 29 base-pair sized fragments, while the 109 base-pair sized fragmentdisappears (track 1: homozygotic; tracks 2 and 3: heterozygotic) (seeFIG. 2).

[0079] Genotype analysis of genomic DNA by restriction digestion.

[0080] Genomic DNA from the tissue samples of the primary tumours wasisolated by standard methods (Current Protocols in Molecular Biology,John Wiley and Sons, Inc., 1995). In order to be able to genotypeanalyse the genomic DNA, the transmembrane region in the FGFR-4 gene wasamplified with the following primers in a PCR reaction:5′-GACCGCAGCAGCGCCCGAGGCCAG-3′ (bp 1129-1142), and5′-AGAGGGAAGAGGGAGAGCTTCTG-3′ (bp 1275-1297). For the PCR reaction,Ready-to-Go PCR Beats (Pharmacia, Uppsala, Sweden) were used. Thefollowing PCR cycles were used: 3 min at 95° C., 45 secs at 94° C., 45secs at 72° C. and 5 mins at 72° C. A total of 35 cycles were performed.The PCR products were incubated for 1 hr at 60° C. with 5 U/25 Wd ofBstN1 (NEB, Schwalbach/Taunus). The DNA fragments from the restrictiondigestion were separated with a 20% polyacrylamide gel and stained withethidium bromide. The ³⁸³Arg allele is characterised by two fragments of80 and 29 bp size, while the ³⁸⁸Gly allele is indicated by a single 109bp sized fragment. Each genotype analysis was repeated three times.

[0081] DNA sequencing of PCR products. For the sequence analysis of thetransmembrane domain of FGFR-4, the PCR products were cloned into theBluescript vector. For this, a PCR reaction was performed as alreadydescribed. The following primers were used: sense-GGGAATTCGACCGCAGCAGCGCCCGAGG; o-sense-GCTCTAGAAGAGGGAAGAGGG AGAG. The PCRproducts of the cloning of FGFR-4^(Arg388)/wt could be directlysequenced in the vector pcDNA3. The DNA sequencing of plasmid DNA wasperformed by the chain termination method. After annealing of theT/-primer onto the plasmid DNA, the sequencing reaction was performedwith T/-DNA polymerase (Pharmacia, Freiburg). The products of thesequencing reaction were then separated on a denaturing 5%polyacrylamide gel (7.5 M urea; 1×TBE) and exposed on Xray film afterdrying (see FIG. 3). From this, the DNA sequences of the wild type andalso of the mutation, were obtained.

[0082] Immunoprecipitation and Western blot analysis. 2.2×10⁶ cells werespread onto 10 cm Petri dishes and incubated overnight. Then the cellmedium was replaced by medium with no foetal bovine serum and incubatedfor a further 24 hrs. For the stimulation, the cells were incubated for10 mins with 50 ng aFGF/ml, washed twice with cold PBS and placed onice. The cells were incubated for 15 mins at 4° C. with 300 pl of coldlysis buffer (1% w/w NP40, 1.25% w/v sodium deoxycholate, 0.1% w/v SDS,0.15 M NaCl 0.01 M sodium phosphate, pH 7.2, 2 mM EDTA, 10 mM sodiumfluoride, InmM PMSF, 20 pg/ml aprotinin, 1 mM orthovanadate, 10 mMsodium pyrophosphate), and the lysate clarified by centrifugation(13,000 RPM) at 4° C. For the protein value determination, the Micro-BCAProtein Assay (Pierce) was used in accordance with the manufacturer'sinstructions. For the immuno-precipitation, the cell lysates wereadjusted to equal protein content and then incubated for 18 hrs at 4° C.with 0.5 μg anti-FGFR-4 (Santa Cruz) and protein-A-Sepharose (PharmaciaFreiburg) on a rotating wheel. The immune complexes were washed 4 timeswith cold HNTG (20 mM HEPES pH 7.5, 150 mM NaCl, 0.1% Triton X100, 10%glycerine, 10 mM sodium pyrophosphate). For sample preparation, theimmune complexes were treated with 50 pi 3×Laemmli buffer and incubatedfor 5 mins at 99° C. The precipitated proteins were separated on a 7.5%SDS-PAGE (see FIG. 1).

[0083] For Western blots, the proteins separated by SDS-PAGE weretransferred to nitrocellulose. Non-specific protein binding sites on themembrane were blocked by incubation for 2 hrs at room temperature withTBS-T/0.25% gelatine (10 mM Tris/HCl pH 8.0, 0.15 M NaCl, 0.05%Tween20). The incubation with primary antibodies was effected for 6 hrsat 4° C. on a tilt snaker. Non-specifically bound antibodies were thenremoved by washing 4 times with TBS-T/0.25% gelatine. The binding ofsecondary antibodies was effected for 1 hr at room temperature. Thenon-specifically bound secondary antibodies were removed by a furtherwashing step. Immune complexes were made visible with the ECLI kit(Amersham, Braunschweig) in accordance with the manufacturer'sinstructions.

[0084] Statistical Methods. Statistical calculations were performed withthe aid of the statistics program MedCalc (MedCalc Software, Belgium)and EpiInfo 6.04b (CDC, Atlanta, Ga.). In order to determine thecorrelations between the genotypes in the different patient groups andthe clinical data, an odds ratio, the confidence interval (CI) and astatistical significance (P value) were calculated. Because of the smallnumber of ³⁸⁸Arg homozygotic patients, this group was combined with thegroup of ³⁸⁸Arg heterozygotic patients for the statistical calculations.

[0085] Detection of FGFR-4 in Tumour Cell Lines. Table 1 shows thecorrelation between the expression of RTK and breast cancer. Expressionof RTK clearly occurs more often in cell lines from breast cancer, whileno expression is detectable in cell lines of normal breast epithelialcells. TABLE 1 Detection of FGFR-4 in Breast Cancer Cells Northern Blot[sic] Breast Cancer Cell Lines FGFR-4  1 HTB-30 (SK-BR-3) ++  2 HTB-122(BT-549) −  3 MCF-7 +  4 BT-483 +++  5 T-47D +  6 ZR-75-1 +++  7MDA-MB-468 −  8 MDA-MB-453 ++++  9 MDA-MB-361 ++++ 10 MDA-MB-415 − 11MDA-MB-231 − Normal Breast Epithelial Cell Lines 12 HBL-100 − 13 MCF-10A−

[0086] From Table 2, it is clear that the G388R mutation also occurs incell lines of other cancer types and is correlatable with these. Inhealthy epithelial cell lines, the mutation is not detectable. TABLE 2Mutation FGFR-4 G388R in various other tumour cell lines Sample genomicDNA cDNA Glioblastoma U-138 −/− −/− U-373 −/− −/− U-172 −/− −/− U-118−/* −/* SF-763 −/− −/− U-1240 */* */* T-98G (*)/− (*)/− U-937 −/− −/−Neuroblastoma SK-N-SH −/* −/* SH-SY-SY −/* −/* Uterine Cancer OAW-42 −/*−/* PA-1 −/− −/− Caov-3 −/− −/− Squamous Hlac-78 −/− −/− Hlac-79 −/− −/−Scc-4 −/− −/− Scc-10a −/− −/− Scc-10b −/− −/− Scc-17a −/− −/− Scc-17b−/− −/− Scc-22a */* */* Scc-22b */* */* HaCat −/− −/− FaDu −/− −/−Normal Breast Epithelial Cell Lines HBL-100 −/− −/− MCF-10A −/− −/−

[0087] Detection of the FGFR-4 Mutation G388R in Biopsies. Table 3 thusshows that of 61 female patients from St Petersburg with breast cancerwho were studied, 56% carried the G388R mutation, 45% of themheterozygotically and 11% homozygotically. Of the 69 female breastcancer patients from Munich who were studied, 43% carried the G388Rmutation, 32% of them heterozygotically and 11% homozygotically. Theproportion of the total percentage of the mutation in female patientsfrom St Petersburg and Munich is different. This suggests that the G388Rmutation is a germ line mutation. TABLE 3 Detection of FGFR-4 mutationG388R in biopsies Samples from breast tumours From St Petersburg FromMunich Sample gen. DNA Sample gen. DNA cDNA 19 102 T */− 5382 T */− */−20 101 N 5609 T */* */* 21 103 T */− 8926 T */− */− 22 103 N */− 9456 T*/* */* 23 2 T */* 9556 T */− */− 24 2 N */* 10347 T −/− −/− 25 12 T −/−10555 T −/− −/− 26 12 N 10681 T */− */− 27 13 T −/− 10781 T */− */− 2813 N 10808 T */* */* 29 14 T */− 11189 T */− */− 30 14 N */− 11526 T */−*/− 31 15 T −/− 11697 T */− 32 15 N 11820 T −/− −/− 33 17 T */− 12015 T−/− −/− 34 17 N */− 12166 T */− */− 35 18 T −/− 13932 T */− */− 36 18 N16003 T */− */− 37 20 T −/− 16353 T −/− −/− 38 20 N 1 N 39 21 T */* 2 T*/− */− 40 21 N */* 3 N 41 22 T */− 4 T −/− −/− 42 22 N 5 N 43 23 T */−6 T −/− −/− 44 23 N 7 N 45 31 T */− 8 T −/− −/− 46 31 N */− 9 N 47 42 T−/− 10 T */− */− 48 42 N 11 N 49 43 T −/− 12 T −/− −/− 50 43 N 13 N 5145 T −/− 14 T */− */− 52 45 N 16 T */− */− 53 47 T */− 17 N −/− 54 47 N*/− 18 T */− 55 48 T −/− 19 N 56 48 N 20 T −/− 57 50 T −/− 38 T −/− 5850 N 3433 T −/− 59 53 T −/− 3539 T 60 53 N 3631 T */* 61 54 T */− 3632 T−/− 62 54 N */− 3636 T −/− 63 55 T −/− 3637 T */− 64 55 N 3638 T −/− 6560 T */− 3640 T −/− 66 60 N */− 991 N −/− 67 61 T */* 991 T −/− 68 61 N*/* 15153 N 69 62 T */− 15153 T −/− 70 62 N */− 15856 N */* 71 63 T */−15856 T */* 72 63 N */− 12845 N 73 67 T */− 12845 T −/− 74 67 N */−19044/93 N 75 69 T −/− 19044/93 T −/− 76 69 N 9426/93 N 77 78 T */−9426/93 T −/− 78 78 N */− 2005 N */− 79 79 T */* 2005 T */− 80 79 N */*14860 N 81 82 T −/− 14860 T −/− 82 82 N 4198 T −/− 83 83 T −/− 5739 T*/* 84 83 N 6060/93 turn */* 85 85 T −/− 6982/93 turn −/− 86 85 N7244/93 turn −/− 87 86 T */− 8114/93 turn −/− 88 86 N */− 8335/93 turn*/− 89 87 T −/− 8481/93 turn */− 90 87 N 8566/93 turn −/− 91 89 T −/−8786/93 turn */* 92 89 N 9145/93 turn −/− 93 94 T −/− 9354/93 turn −/−94 94 N 9796/93 turn −/− 95 97 T */* 9798/93 turn −/− 96 97 N */−10125/93 turn −/− 97 98 T −/− 10150/93 turn */− 98 98 N 11218/93 turn−/− 99 99 T */* 11673/93 turn −/− 100 99 N */* 13232/93 turn −/− 101 100T −/− 13316/93 turn */− 102 100 N 14724/93 turn −/− 103 101 T */−14879/93 turn #1 −/− 104 101 N */− 14879/93 turn #2 −/− 105 102 T (#20)*/− 15645/93 turn −/− 106 102 N (#19) */* 107 103 T (#22) */− 108 103 N(#21) 109 104 T */− 110 92 T */* 111 65 T 112 52 T */− 113 35 T */− 11433 “A” T */− 115 33 “B” mts. */− 116 30 T */− 134 30 N */− 117 27 T */*133 27 N */* 118 24 T */− 119 10 T */− 132 10 T */− 120 3 T −/− 121 90 T−/− 122 90 N 123 80 T −/− 124 80 N 125 81 T −/− 126 58 T −/− 127 51 T*/− 128 51 N 129 44 T */− 130 44 N

[0088] Correlation between the FGFR-4-G388R mutation and the detectionof FGFR-4 expression. From Table 4 below, it is clear that the G388Rmutation (genomic DNA and cDNA) only occurs when expression and/orintensified expression occurs. The mutation is detectable neither in thenormal breast epithelial cell tines nor in the breast cancer cell linesin which no RTK expression was found.

[0089] The cell line MDA-NM 453, whose RTK expression is especiallypronounced, shows a homozygotic G388R mutation. TABLE 4 Correlationbetween the FGFR-4-G388R mutation and the detection of FGFR-4 expressionNorthern Blot Mutation Breast cancer cell line FGFR-4 cDNA gen. DNA  1HTB-30 (SK-BR-3) ++ +/+ +/+  2 HTB-122 (BT-549) − −/− −/−  3 MCF-7 + +/−+/−  4 BT-483 +++ +/− +/−  5 T-47D + +/− +/−  6 ZR-75-1 +++ +/− +/−  7MDA-MB-468 − −/− −/−  8 MZDA-MB-453 ++++ +/+ +/+  9 MDA-MB-361 ++++ +/−+/− 10 MDA-MB-415 − −/− −/− 11 MDA-MB-231 − +/− +/− Normal BreastEpithelial Cell Lines 12 HBL-100 − −/− −/− 13 MCF-10A − −/− −/−

[0090] Study of the Correlation Between the Occurrence of the FGFR-4Mutation G388R and Lymph Node Metastasis Status or Relapse-Free SurvivalTime

[0091] Table 5 shows the clinical parameters of all patients who tookpart in the study of the role of the G388R mutation in the tumorigenesisof breast cancer. It is found that patients with a G388R mutation have aworse long-term prognosis than patients with no G388R mutation.

[0092] Key to Table 5: [on Following Pages]

[0093] Her2: expression level of the Her2 receptor; 0=no expression to3=overexpression

[0094] OPDAT: date of the operation

[0095] M/R: metastasis formation/relapse; 0=no/1=yes

[0096] Vers: died; 0=no/I=yes

[0097] UBERRE: survival time without relapse, in months

[0098] Grade: differentiation grade of tumour; 1=strongdifferentiation/3=low differentiation stage: size of the primary tumour.

[0099] E-Rec: expression of the oestrogen receptor; 0=no expression to12=highest expression

[0100] GEN: genotype of the FGFR-4; G=wild type allele; R=mutated allele

[0101] BEDAT: date of last observation

[0102] REZDAT: date of relapse diagnosis

[0103] TODDAT: date of death

[0104] UBERLEB: survival time overall

[0105] Nod.: metastases in the lymph nodes; 0=no/I=yes

[0106] Men: menopause

[0107] P-Rec: expression of the progesterone receptor: 0=no expressionto 12=highest expression TABLE 5 FGFR-4 genotypes and clinical case dataPathoNr. Her2 GEN OPOAT BEDAT M/R REZDAT Vers TODDAT ÜBERRE Überleb489292 G/G 31.03.92 14.02.97 0 0 59 59 617792 G/G 24.04.92 14.07.94 0 027 27 639792 0 G/G 29.04.92 03.04.94 0 0 23 23 724493 G/G 11.05.9323.06.97 0 0 48 48 914593 0 G/G 16.06.93 07.03.96 0 0 33 33 935493 2+G/G 21.06.93 15.03.96 0 0 33 33 963392 G/G 30.06.92 30.06.97 0 0 60 60979693 G/G 29.06.93 07.03.96 0 0 33 33 979893 1+ G/G 29.06.93 30.06.97 00 48 48 1034792 0 G/G 13/07.92 26.03.97 0 0 56 56 1323293 G/G 02.03.9305.08.97 0 0 48 48 1331693 3+ G/G 30.09.92 29.03.95 0 0 36 36 1564593 1+G/G 19.10.92 19.06.97 0 0 56 56 78692 G/G 19.01.88 29.06.93 0 0 65 65176989 3+ G/G 0 0 84 84 273690 1+ G/G 0 0 72 72 725289 G/G 0 729991 0G/G 0 0 57 57 733290 1+ G/G 0 1 28.03.87 14 14 826790 2+ G/G 0 0 91 91867191 0 G/G 0 0 79 79 988590 G/G 0 0 90 90 991790 0 G/G 0 0 68 681031190 2+ G/G 0 0 90 90 1033391 1+ G/G 0 0 55 55 1055592 G/G 0 025.04.93 57 79 1101191 0 G/G 0 0 70 70 1426491 1+ G/G 0 0 76 76 1560492G/G 0 0 42 42 1605790 0 G/G 0 0 63 63 1641488 2+ G/G 0 0 109 109 11218931+ G/G 23.07.93 18.01.96 1 06.09.94 1 18.01.96 14 16 1201592 1+ G/G10.08.92 25.04.95 1 25.04.95 0 29 29 258093 0 G/G 22.02.85 18.04.91 130.11.89 57 102 479090 3+ G/G 04.04.86 23.07.89 1 12.04.88 1 23.07.89 2455 806490 0 G/G 14.06.86 18.09.90 1 10.11.88 1 18.09.90 29 71 963589 1+G/G 26.07.85 06.03.89 1 26.04.88 1 06.03.89 33 60 972291 3+ G/G 09.07.8719.08.89 1 16.07.88 1 19.08.89 12 35 995589 0 G/G 01.08.85 27.06.88 129.04.87 1 27.06.88 21 40 1112389 3+ G/G 30.08.85 14.07.86 1 14.06.86 114.07.86 9 14 1726892 G/G 18.11.88 23.04.91 1 07.11.90 1 23.04.91 24 40289791 3+ G/R 25.02.87 02.04.92 0 0 61 61 337293 G/R 03.03.89 04.03.92 00 36 36 879290 2+ G/R 01.07.86 01.10.92 0 1 01.10.92 75 103 893090 0 G/R03.07.86 17.12.93 0 0 89 89 1106192 G/R 23.07.88 04.02.94 0 0 66 661107789 G/R 29.08.85 13.02.92 0 0 77 77 1113892 G/R 24.07.92 14.10.97 00 63 63 1118990 G/R 19.08.86 15.02.92 0 0 66 66 1152692 3+ G/R 31.07.9230.06.97 0 0 59 59 1599789 1+ G/R 14.12.85 15.02.87 0 1 15.02.87 14 191614591 0 G/R 12.11.87 25.02.92 0 0 51 51 92390 0 G/R 20.01.86 13.02.921 09.02.91 0 61 73 99289 0 G/R 23.01.85 17.03.92 1 22.02.90 0 61 86130588 2+ G/R 20.01.84 30.01.92 1 18.02.87 0 37 96 306490 3+ G/R01.03.86 06.06.87 1 06.11.86 1 06.06.87 8 21 492191 3+ G/R 07.04.8720.04.94 1 10.10.90 0 42 84 529692 G/R 07.04.92 29.03.96 1 29.03.96 3446 529990 3+ G/R 17.04.86 29.04.87 1 14.04.87 1 29.04.87 12 17 538292 3+G/R 07.04.92 13.10.93 1 07.04.92 1 13.10.93 0 18 614091 2+ G/R 29.04.8721.04.90 1 31.07.88 1 21.04.90 15 49 651591 0 G/R 07.05.87 17.09.93 114.04.90 0 35 76 673592 G/R 06.05.92 27.02.96 1 06.06.93 0 13 45 6780922+ G/R 07.05.92 29.06.96 1 07.05.92 0 0 49 714289 2+ G/R 04.06.8516.09.87 1 30.06.87 1 16.09.89 25 71 807492 0 G/R 29.05.92 29.11.96 129.11.96 0 54 54 848193 G/R 03.06.93 10.09.94 1 16.08.94 1 10.09.94 1516 955692 1+ G/R 29.06.92 26.11.96 1 17.08.95 37 53 1022090 0 G/R29.07.86 02.07.88 1 23.06.88 1 02.07.88 23 32 1054987 G/R 05.08.8315.12.84 1 29.02.84 1 15.12.84 7 23 1078192 2+ G/R 20.07.92 22.10.95 125.09.95 0 38 39 1079689 2+ G/R 22.08.85 27.03.91 1 31.12.89 1 27.03.9152 93 1169792 2+ G/R 04.08.92 01.07.95 1 01.07.95 1 01.07.95 47 471216692 0 G/R 12.08.92 30.07.96 1 09.09.94 0 23 48 1314689 2+ G/R16.10.85 21.02.92 1 05.11.91 0 73 76 1391992 2+ G/R 17.09.92 01.05.93 115.01.93 1 15.01.93 5 8 1696290 G/R 11.12.86 20.02.92 1 30.11.89 0 36 62920891 G/R 213593 R/R 10.02.89 20.04.94 0 0 62 62 313791 3+ R/R 28.02.8721.02.92 0 0 60 60 878693 R/R 09.06.93 07.09.96 0 0 33 33 1107391 3+ R/R01.08.87 29.02.92 0 0 55 55 1125690 R/R 20.08.86 13.10.93 0 0 86 86120788 R/R 27.01.84 24.04.86 1 24.04.86 24 37 560992 3+ R/R 13.04.9204.04.93 1 10.04.92 1  4.4.93 0 12 1008692 2+ R/R 08.07.92 11.07.94 117.08.93 1 11.07.94 13 24 1686490 3+ R/R 10.12.86 23.07.87 1 07.05.87 123.07.87 5 10 PathoNr. Age slage Nod. Grade Men E-Rec P-Rec 489292 71.71c 0 2 2 12 9 617792 1 639792 0 724493 64.3 2 1 2 2 12 12 914593 86.2 22 2 8 9 935493 49.6 2 1 3 2 0 0 963392 79.4 1b 0 2 2 3 12 979693 46.7 20 2 3 9 0 979893 77 4b 1 2 2 1 0 1034792 43.5 3 1 2 1 0 6 1323293 61.4 21 3 2 0 0 1331693 71.2 2 1 2 2 6 1 1564593 65.3 2 1 2 2 0 0 78692 63.8 20 3 2 4 12 176989 43.4 2 1 2 1 273690 52.3 2 0 2 1 12 12 725289 49.5 2 02 0 0 729991 65.7 2 1 2 2 12 12 733290 81.7 1b 0 2 2 12 12 826790 77.7 20 2 2 9 0 867191 77.5 2 0 3 2 0 0 988590 61.5 1b 0 2 2 6 9 991790 50.71c 0 2 2 8 4 1031190 43.4 1c 0 2 1 1033391 62.4 2 1 3 2 12 12 105559277.8 2 0 2 2 6 12 1101191 48.5 2 0 2 1 0 0 1426491 54.9 1b 0 3 2 0 01560492 50 1c 0 3 3 0 0 1605790 56.4 1c 1 2 2 12 6 1641488 67.4 1b 0 2 29 1121893 59.9 2 1 3 2 4 2 1201592 48.9 1c 1 2 1 1 6 258093 79.4 1c 1 22 479090 67.9 1c 0 2 2 0 0 806490 66.7 2 1 2 2 8 0 963589 47.8 2 1 3 1 0972291 48.3 2 0 3 1 0 0 995589 58.4 3 1 3 2 6 1112389 79.4 4b 2 2 2 0 01726892 38.7 2 0 3 1 0 0 289791 48.1 2 1 3 2 0 0 337293 51.2 1c 0 2 1 62 879290 44.9 1c 0 3 1 1 6 893090 68.5 4 0 3 2 12 6 1106192 53.9 1c 0 22 2 12 1107789 50.3 1c 0 2 1 8 12 1113892 51.9 2 0 1 2 2 6 1118990 53.91c 0 2 2 6 4 1152692 57.7 2 1 2 2 4 12 1599789 80.2 1c 0 3 2 3 2 161459176.7 2 1 2 2 3 9 92390 45.8 2 1 3 1 0 1 99289 47.1 1c 1 2 1 130588 39 21 2 1 306490 52.2 2 1 3 3 0 0 492191 56.2 2 1 3 2 3 0 529692 66.9 1b 0 32 0 0 529990 77.1 2 1 3 2 0 0 538292 56.1 4 2 3 2 0 0 614091 47.2 2 2 21 2 6 651591 49.3 3 1 2 1 3 9 673592 36.8 2 0 2 1 0 0 678092 56.7 1c 1 22 0 0 714289 55 1c 1 2 2 0 807492 1 848193 58 2 2 2 2 0 4 955692 49.9 1c1 3 3 2 1 1022090 51.7 2 2 2 2 12 1 1054987 71.5 2 0 3 2 1078192 79.4 21 3 2 1 9 1079689 81.4 4 2 3 2 1169792 67.3 2 1 3 2 6 9 1216692 45.3 2 12 1 4 6 1314689 81.2 4 1 2 2 6 12 1391992 76 2 1 3 2 1696290 55.2 2 0 22 6 6 920891 50.5 2 0 3 1 2 6 213593 45.3 1c 0 2 1 3 6 313791 68.6 2 1 32 0 0 878693 0 1107391 1c 0 2 2 3 1 1125690 43.1 3 1 3 1 0 0 120788 68.12 1 3 2 560992 59.1 4b 2 3 2 6 0 1008692 47.4 2 1 3 2 0 0 1686490 55.4 21 3 2 0 0

[0108] From FIG. 4, it is clear that the G388R mutation is to be foundin greater number in patients who already have metastases in the lymphnodes at the time of the first treatment. Of the patients with a G388Rmutation, 62.7% had lymph node metastases, while of the patients with noG388R mutation only 38.2% displayed metastases in the lymph nodes. Asthe lymph node metastasis status is an important prognostic marker forthe further discrimination of tumours with a worse and those with abetter prognosis, it can be concluded from this result that the G388Rmutation in the 85 patients studied leads to a more severe tumourprogression.

[0109] From FIG. 5 it is can be seen that in the group of patientsstudied, the relapse-free survival probability is very much lower forthose with a G388R mutation than for those patients who have no G388Rmutation. While 74.4% of the patients with a relapse possess the 388Rgenotype, only 25.6% have the 388G genotype. This shows that patientswith the G388R mutation suffer a relapse more quickly, and thereforecould not be successfully treated.

[0110] In summary, it can be stated that the FGFR-4 mutation G388R leadsto a 2.7-fold (OR=2.7; CI: 1.02<OR<7.4) increased risk of metastasisformation in the lymph nodes and to a 5.44-fold (OR=5.44; CI:1.93<OR<7.4) increased risk of a tumour relapse. Patients with a mutatedFGFR-4 allele (G388R) thus seem to have a predisposition to a tumourrelapse and hence a poorer disease prognosis. Materials AcrylamideServa, Heidelberg Agar Difco, Detroit Agarose BRL, Eggenstein AmpicillinBoehringer, Mannheim Aprotinin Sigma, Taufkirchen N,N′-bisacrylamideRoth, Karlsruhe Caesium chloride BRL, Eggenstein DesoxynucleotidesPharmacia, Freiburg Ethidium bromide Sigma, Taufkirchen Gelatine Sigma,Taufkirchen Guanidium isotbiocyanate Fluka, Switzerland HEPES Serva,Heidelberg Sodium fluoride Sigma, Taufkirchen PMSF Sigma, TaufkirchenSDS Roth, Karlsruhe Tris Riedel de Haen, Seelze Triton X100 Serva,Heidelberg Tween20 Sigma, Taufkirchen

[0111] All substances not listed here came from the firms Sigma(Tauflirchen), Serva (Heidelberg), Riedel De Haen or Merck (Darmstadt)and the highest possible purity grades were used. InstrumentsElectrophoresis of DNA Workshop, MPI for Biochemistry, MartinsriedElectrophoresis of proteins Atto, Japan Refrigerated centrifuge Heraeus,Hanau Biofuge 17S Protein transfer Semidry blot apparatus, Workshop, MPIfor Biochemistry, Martinsried Sterile workbench Biogard, The BakerCompany, USA Cell culture Incubator B5060 EX/CO₂, Heraeus, Hanau Cellcounting Coulter Counter, Coulter Electronics, Glasgow.

LITERATURE

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[0121] Thompson J A, Haudenschild C C, Anderson K D, DiPietro J M,Anderson W F and Maciag T. (1989): Proc. Natl. Acad. Sci. USA, 86,7928-7932 (1989). Cell Line Origin No. U-138 ATTC HTB-16 U-373 ATTCHTB-17 U-172 U-118 ATTC HTB-15 SF-763 SUGEN U-1240 SUGEN T-98G SUGENU-937 ATCC CRL-1593 SK-N-SH ATCC HTB-11 SH-SYSY F. J. Klinz OAW-42 DKFZPA-1 ATCC CRL-1572 Caov-3 ATCC HTB-75 Hlac-78 Dr. Wustrow Hlac-79 Dr.Wustrow Scc-4 ATCC CRL-1624 Scc10a Dr. Wustrow Scc10b Dr. Wustrow Scc22aDr. Wustrow Scc22b Dr. Wustrow Scc17a Dr. Wustrow Scc17b Dr. WustrowFaDu Dr. Wustrow HaCat HBL100 ATCC HTB-124 MCF10A ATCC CRL-10317 SKBr-3ATCC HTB-30 BT-549 ATCC HTB-122 MCF-7 ATCC HTB-22 BT483 ATCC HTB-121T-47-0 ATCC HTB-133 ZR-75-1 ATCC CRL-1500 MDA-MB-468 ATCC HTB-132MDA-MB-453 ATCC HTB-131 MDA-MB-361 ATCC HTB-27 MDA-MB-415 ATCC HTB-128MDA-MB-231 ATCC HTB-26 K-562 ATCC CCL-243

[0122]

1 8 1 25 PRT Homo sapiens DOMAIN (1)..(25) amino acid sequence of FGFR-4(mutant) between positions 366-390 1 Arg Tyr Thr Asp Ile Ile Leu Tyr AlaSer Gly Ser Leu Ala Leu Ala 1 5 10 15 Val Leu Leu Leu Leu Ala Arg LeuTyr 20 25 2 25 PRT Homo sapiens DOMAIN (1)..(25) amino acid sequence ofFGFR-4 (wild-type) between positions 366-390 2 Arg Tyr Thr Asp Ile IleLeu Tyr Ala Ser Gly Ser Leu Ala Leu Ala 1 5 10 15 Val Leu Leu Leu LeuAla Gly Leu Tyr 20 25 3 29 DNA artificial sequence PCR primer for theamplification of FGFR-4 (wild-type and mutant) 3 gctcagaggg cgggcgggggtgccggccg 29 4 33 DNA artificial sequence PCR primer for theamplification of FGFR-4 (wild-type and mutant) 4 ccgctcgagt gcctgcacagccttgagcct tgc 33 5 24 DNA artificial sequence PCR primer for theamplification of the transmembrane domain of FGFR-4 (wild-type andmutant) 5 gaccgcagca gcgcccgagg ccag 24 6 23 DNA artificial sequence PCRprimer for the amplification of the transmembrane domain of FGFR-4(wild-type and mutant) 6 agagggaaga gggagagctt ctg 23 7 28 DNAartificial sequence primer for sequencing of the transmembrane domain ofFGFR-4 (wild-type and mutant) 7 gggaattcga ccgcagcagc gcccgagg 28 8 25DNA artificial sequence primer for sequencing of the transmembranedomain of FGFR-4 (wild-type and mutant) 8 gctctagaag agggaagagg gagag 25

1. A method for the prophylactic and/or therapeutic treatment of areceptor tyrosine kinase (RTK)-hyperfunction-induced disorder in amammal, which method comprises administering to a mammal in need thereofan effective amount of at least one inhibitor of fibroblast growthfactor receptor-4 (FGFR-4), wherein said RTK-hyperfunction-induceddisorder is treated prophylactically and/or therapeutically.
 2. Themethod of claim 1, wherein said RTK-hyperfunction-induced disorder isone or more disorders selected from the group consisting of cancer, adisease attributable to cellular hyperproliferation and/or cellularinvasion of tissue, a carcinoma, and a metastasis.
 3. The method ofclaim 2, wherein said disorder is breast cancer, squamous cellcarcinoma, glioblastoma, neuroblastoma or uterine cancer.
 4. The methodof claim 1, wherein said inhibitor is a kinase-inactive receptor.
 5. Themethod of claim 1, wherein an overexpression and/or an altered activityof FGFR-4 is lowered and/or inhibited.
 6. The method of claim 5, whereinthe overexpression and/or the altered activity of FGFR-4 is triggered bya mutation of the FGFR-4.
 7. The method of claim 6, wherein the mutationis one or several point mutations.
 8. The method of claim 7, wherein theone or several point mutations leads to an exchange of a hydrophobicamino acid for a hydrophilic amino acid.
 9. The method of claim 6,wherein the mutation occurs in the transmembrane domain of FGFR-4. 10.The method of claim 9, wherein the mutation is one or several pointmutations that lead to an exchange of a hydrophobic amino acid for ahydrophilic amino acid.
 11. The method of claim 6, wherein the mutationoccurs at amino acid position 388 in the FGFR-4 molecule.
 12. The methodof claim 11, wherein the mutation leads to an exchange of glycine forarginine.
 13. The method of claim 6, wherein the mutation is a germ linemutation.
 14. The method of claim 1, wherein the FGFR-4 is mutated andthe inhibitor inhibits the mutated FGFR-4.
 15. A method of diagnosing anRTK-hyperfunction-induced disorder or a genetic predisposition thereforin a mammal, which method comprises determining the presence of amutated FGFR-4 protein or a nucleic acid encoding a mutated FGFR-4protein in a sample of protein or nucleic acid, respectively, obtainedfrom said mammal, wherein the presence of such a protein or nucleic acidis indicative of an RTK-hyperfunction-induced disorder or a geneticpredisposition therefor.
 16. The method of claim 15, wherein saidRTK-hyperfunction-induced disorder is cancer.
 17. The method of claim15, which method comprises contacting the sample of nucleic acid with alabeled DNA or RNA molecule encoding a mutated FGFR-4 under hybridizingconditions and detecting the labeled DNA or RNA molecule afterhybridization, wherein the detection of the labeled DNA or RNA isindicative of the presence of a nucleic acid molecule encoding a mutatedFGFR-4 in the sample.
 18. The method of claim 15, which method comprisescontacting the sample of nucleic acid with a restriction enzyme whoserecognition sequence is affected by the mutation in the mutated FGFR-4and detecting the presence or absence of fragments or the presence ofaltered fragments of the nucleic acid after contact with the restrictionenzyme, wherein the absence of fragments or the presence of alteredfragments of the nucleic acid after contact with the restriction enzymeis indicative of the presence of a nucleic acid molecule encoding amutated FGFR-4 in the sample.
 19. The method of claim 18, wherein themutation in the mutated FGFR-4 occurs at amino acid position
 388. 20. Amethod of identifying an inhibitor of a mutant FGFR-4, which methodcomprises contacting a potential inhibitor with a mutated FGFR-4 anddetermining activity in the absence and presence of the potentialinhibitor, wherein a decrease in activity in the presence of thepotential inhibitor indicates that the potential inhibitor is aninhibitor of mutant FGFR-4.
 21. The method of claim 20, wherein theactivity is tyrosine kinase activity.
 22. The method of claim 20,wherein the activity is interaction with a protein with which FGFR-4interacts.
 23. A method for the prophylactic and/or therapeutictreatment of a RTK-hyperfunction-induced disorder in a mammal, whichmethod comprises administering to a mammal in need thereof an effectiveamount of at least one inhibitor of a mutant FGFR-4 identified inaccordance with the method of claim 20, wherein saidRTK-hyperfunction-induced disorder is treated prophylactically and/ortherapeutically.
 24. A method for the prophylactic and/or therapeutictreatment of a RTK-hyperfunction-induced disorder in a mammal, whichmethod comprises administering to a mammal in need thereof an effectiveamount of at least one inhibitor of a mutant FGFR-4 identified inaccordance with the method of claim 21, wherein saidRTK-hyperfunction-induced disorder is treated prophylactically and/ortherapeutically.
 25. A method for the prophylactic and/or therapeutictreatment of a RTK-hyperfunction-induced disorder in a mammal, whichmethod comprises administering to a mammal in need thereof an effectiveamount of at least one inhibitor of a mutant FGFR-4 identified inaccordance with the method of claim 22, wherein saidRTK-hyperfunction-induced disorder is treated prophylactically and/ortherapeutically.
 26. An antibody that reacts specifically with a mutatedFGFR-4 receptor, which is overexpressed and/or has altered activity in acell.
 27. The antibody of claim 26, wherein a hydrophobic amino acid inthe wild-type receptor has been exchanged for a hydrophobic amino acidin the mutated FGFR-4 receptor.
 28. The antibody of claim 26, whereinthe transmembrane domain of the mutated FGFR-4 receptor comprises apoint mutation.
 29. The antibody of claim 28, wherein the point mutationoccurs at amino acid 388 and, optionally, results in replacement ofglycine with arginine.